P381: DNA METHYLATION PROFILING OF MESENCHYMAL STROMAL CELLS ISOLATED FROM FEMURAL HEAD BONE MARROW VERSUS BONE MARROW ASPIRATES: RELEVANCE FOR AML STUDY BASED CONTROLS
Authors: A. Abdul-Aziz, C. Weigel, A. Kovacs, Y.-Z. Wu, J. Byrd, E. Hertlein, C. Oakes
Journal: HemaSphere (2022)

Background: Bone marrow mesenchymal stromal cells (BMSCs) are important precursors for multiple differentiated cell types. Alterations in BMSCs contribute to several disease states, including acute myeloid leukemia (AML) and other hematologic cancers. As BMSCs are used as controls for research and obtained from various bone tissue locations with active hematopoiesis, it is important to understand the developmental capacity and potential differential phenotypes of BMSCs derived from various anatomical sites. Epigenetic regulation plays an essential role in cell lineage specification and development; carefully controlled transcription factor (TF) activity and other chromatin-associated proteins modify the genomic landscape to generate stable, cell type-specific global patterns of gene expression. Aims: We examined genome-wide DNA methylation patterns in BMSCs derived from either femoral head bone marrow or bone marrow aspirates of the iliac crest of healthy donors to identify the optimal control cell population for hematological malignancy research. Methods: Hip-derived BMSCs were obtained from femur heads obtained during hip replacement surgeries (n=19). All patients provided informed consent in accordance with an IRB-approved protocol and the Declaration of Helsinki. Aspirate-derived BMSCs (n=7) were expanded from bone marrow mononuclear cells obtained from a commercial source (AllCells). For genome-wide DNA methylation analyses, the EPIC/850K array platform was used following manufacturer’s protocols (Illumina). Analyses included data obtained from the Gene Expression Omnibus (GEO, GSE79695 and GSE124390) generated from 450K (n= 12) and 850K array data (n=10), respectively. Overlapping probes between the arrays that passed QC were used for downstream analysis. Sex chromosomes and non-CpG probes were removed. The data was reduced to 5,000 most-variable CpGs followed by clustering analysis as described in results. Results: Unsupervised clustering using principal component analysis (PCA) and hierarchical clustering revealed two groups that segregated perfectly between hip-derived versus aspirate-derived BMSCs. There were no observable differences in aspirate-BMSCs generated by our group and those obtained from GEO. Two group supervised analysis (hip versus aspirate) revealed 4,568 CpGs (probes) hypomethylated in hip-derived samples (q<0.05, difference>20%) and 1,433 probes hypermethylated in hip-derived samples (q<0.05, difference>20%). DNA sequence motif enrichment analysis using the HOMER software revealed that hypomethylation in hip-derived BMSCs involved activation of CRE, HOX and AP-1 family TFs. We also observed parallel hypomethylation of the promoters of CREB5, genes from the HOXB and HOXC clusters, and several genes of the MAPK pathway. Hypermethylation in hip-derived BMSCs conversely involved RUNX motifs, corresponding with hypermethylation of the RUNX3 gene. We also observed robust DNA methylation changes at the promoters of other lineage-specifying genes including TBX3, TBX5, PITX2, NR2F2, and EN1. Pathway analysis of genes demonstrating promoter hypomethylation in hip-derived samples revealed strong enrichment for adipocyte progenitors and development. Summary/Conclusion: These findings demonstrate that hip-derived BMSCs are phenotypically distinct from aspirate-derived BMSCs, which may affect their potential use in both therapeutic and research settings where stem-like non-primed BMSCs are required. Our study highlights the importance of using proper selection of control cells to study the role of BMSCs in hematologic malignancies.